Method and system for preparing soft tissue for grafting, enhancing grafting results, and grafting autologous fat and adipocyte derived stem cells to soft tissue such as the breast and other tissue defects

ABSTRACT

A method is disclosed for preparing a soft tissue site, and augmenting the soft tissue site, such as the breast(s), scar, depression, or other defect, of a subject through use of devices that exert a distractive force on the breast(s) and grafting of autologous fat tissue such as domes with sealing rims for surrounding each of the soft tissue site and a regulated pump. The method for preparing the soft tissue site, and enhancing fat graft results, entails application of the distracting force to the targeted soft tissue site at least intermittently for some period of time and preferably several weeks prior to the graft procedure. A related aspect of the invention includes following the preparation steps by transfer of fat from other areas of the subject to the subject&#39;s soft tissue site, and then reapplication of the distractive force to the soft tissue site that received the autologous fat graft. Alternatively, fat from genetically related sources may be used, and the fat may be further processed prior to injection. Substantial soft tissue augmentation, high rates of graft survival and negligible graft necrosis (data demonstrating 80% survival and only 20% necrosis is presented) or calcification result from the practice of these methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods of preparing soft tissue,such as breasts, for augmentation and, relatedly, to methods ofenhancing fat and adipocyte derived stem cells grafting results, andaugmenting and restoring soft tissue by autologous fat and adipocytederived stem cell grafting.

2. Related Art

There are numerous instances where persons desire augmentation of softtissue such as their breasts. Other examples of soft tissue augmentationopportunities include the face, the buttocks, depressed scar contours,tissue atrophy related to aging, scarring, radiation or disease, or anybody deformity or area that is desirably augmented. Another suchinstance is for the augmentation or the restoration of one or bothbreasts subjected to a partial or total mastectomy, a biopsy, or otherdisfiguring events in order to restore physiological symmetry andpsychological well-being. Yet other instances are for correction ofnatural abnormalities such as scarring and dimpling. Still otherinstances are for augmentation of the breasts to improve cosmetics andself-esteem.

Liposuctioned fat is known to be rich in stem cells that have theability to restore and reconstruct various soft tissue defects inresponse to local differentiation clues from the recipient site. Thisinvention also relates to providing for improved survival of largergraft volumes in a given recipient site.

Prosthetic implants have been developed for insertion below the skin.However, the severity of the potential complications including scarring,implant rupture, capsular contracture, necrosis and implant migration aswell as the recent adverse publicity thereof have significantly reducedthe desirability of these implants. While scientific evidence is notconclusive, there is some indication that breast implants mightcontribute to secondary diseases. Thus, there is a societal need forother means to obtain breast augmentation.

Breast augmentation by use of fat grafts was experimentally explored inthe past but has not gained widespread acceptance. First, the largerblocks of grafts used then displayed poor survival. Secondly, the failedgrafts sometimes produce stellate and irregular nodules withcalcifications. These nodules, which were often palpable, wereoccasionally indistinguishable from cancer with the breast imagingtechnology available at that time. For these reasons certain medicalsocieties and associations advocated a ban on fat grafting to thebreast. Consequently, the practice of fat grafting to the breast wascontraindicated and not pursued.

Subsequently, digital mammography, high-resolution ultrasound, MagneticResonance Imaging (MRI) and its associated dynamic absorption curve andfat suppression views have revolutionized breast imaging. As a result,radiologists now have the imaging technology that enables them todifferentiate with reasonable certainty a neoplastic process from thefat necrosis commonly left behind following reduction mammoplasties, thenow accepted SAL (Suction Assisted Lipectomy) breast reduction methods,breast biopsies, and even non-surgery related every day breast trauma.It is consequently evident that concerns associated with distinguishingareas of fat necrosis resulting from fat grafting to the breast fromareas of neoplasia have now been essentially obviated by advances inimaging technology. With these advances, fat grafting may now again beconsidered for this application.

Described herein is a new method of preparing soft tissue foraugmentation, including preferably the breasts, for enhancing fatgrafting results in soft tissue, and augmenting soft tissue by use ofautologous fat grafts. This may preferably be a general method forpreparing a soft tissue recipient to successfully accept the survival oflarger graft volumes, whether these are fat droplets, stem cells orother dispersed live cell injections. This method does not suffer fromthe defects of the previously attempted methods of fat grafting to thebreast in that it results in high rates of larger volume graft survivaland marked reductions of graft necrosis and calcification. This methodis distinguished from previously disclosed methods by, inter alia, theuse of devices that exert a distractive force on the breast to increasethe volume of the recipient space, increase its vascularity and generatea fertile extracellular fluid matrix which prepares the soft tissue sitefor improved graft survival. This technique enhances the results inconjunction with improved techniques of autologous fat harvesting,processing and grafting and other techniques of tissue culture.

Soft tissue grafting, augmentation and restoration of tissue loss bydispersion of small micro-grafts is three dimensional grafting whichrequires novel concepts. Just as in two dimensional grafting, one cannotapply more grafts than the surface area of the defect, and stacking moregrafts is counterproductive. In 3D grafting, even with the best and mosteven dispersion of the finest graft droplets, one cannot graft more thanthe volume of the recipient site defect (see drawing figure). It followstherefore that expansion, even temporarily, of the recipient space byexternal distraction would increase its volume and create room for moregrafts to survive. In addition, it is well known that tissue expansionis angiogenic, (increases the vascularity) and that improved vascularityleads to more successful engraftment. Furthermore, the increasedextracellular fluid matrix of the expanded tissue is rich in growthfactors and other agents known to render the site more fertile for graftsurvival.

SUMMARY OF THE INVENTION

In addition to the foregoing, the invention relates to a method ofaugmenting soft tissue of the type mentioned herein, includingpreferably at least one breast of a subject, comprising the steps ofapplying a distractive force to at least one breast of a subject,removing a sample of fat from another body area of the subject,injecting at least a portion of said sample of fat taken from thesubject into said breast that was subjected to a distractive force andthen possibly maintaining the distractive force to the breast injectedwith the subject's fat to immobilize the recipient tissue and help withthe engraftment. Use of this method results in substantial augmentationof the subject's breast or breasts while also serving to remove fat fromundesirable locations of the body such as the abdomen, thighs, flanks,buttocks (gluteal regions), and trochanters. Use of an autologous graftalso reduces the likelihood of graft rejection. The subject may be ahuman or more particularly a female human.

The invention further relates to preparing a soft tissue site, includingpreferably the breasts, for augmentation or regeneration of tissue lossto make it more receptive to successful engraftment of liposuctionedfat, adipocyte derived stem cells, or other dispersed tissue graftdroplets, through pre-grafting expansion of the soft tissue site.

In practicing a preferred embodiment of the method of this invention, adistractive force is applied to the breast both before and possiblyafter grafting of fat to the breast. The distractive force may beapplied via any number of different methods or devices, both externallyand internally. A preferred method for applying a distractive forcecomprises the application of vacuum pressure to the breast of saidsubject by use of a device comprising a dome which fits over thefat-receiving breast(s) of the subject, a rim that seals said dome(s)around said breast(s), and a pump for creating a vacuum within thedome(s) around said breast. An external breast tissue expander (theBrava® Breast Enhancement and Shaping System, Brava, LLC; Coconut Grove,Fla.) represents one commercially available and preferred device forapplying vacuum pressure to the breast of the subject. Vacuum pressuremay be applied to the breast or breasts by a hand pump that may bedetachable or by a self-contained vacuum pump system that is connectedto the device. This self contained vacuum pump system may comprise anindependent power source, a pressure sensor, and a servomechanism tomaintain a constant vacuum pressure inside said dome. Under certaincircumstances, equivalent vacuum pressure may be applied to both breastsin the practice of the invention. However, other circumstances maydictate that different vacuum pressures are applied to each breast, orto only the fat-receiving breast.

The fat sample used in the breast graft is preferably typically removedfrom at least one of an abdominal region, a thigh region, a flankregion, a gluteal region, or a trochanter of said subject. However, thesample of fat may be taken from any suitable region of the subject.Removal of fat is desirably effected by methods that minimize trauma andminimize manipulation of the fat removed from the subject. One suitablemethod of obtaining the sample of fat is by aspirating the fat from thesubject with a syringe. This syringe may be a 10, 20 or 60 ml syringefitted with a 12 or 14 gauge cannula (i.e. approximately 2-3 mm indiameter). The harvested fat is then separated from any fluid such asserum or tumescent fluid that is associated with the fat. These fatremoval and separation methods are well known in the medical art.

In an alternative embodiment, the aspirated tissue is processed toconcentrate, multiply or increase the number of stem cells present usingtechniques that are familiar to those of ordinary skill in the art.

The fat or the stem cell sample is preferably then injected into thebreast that was previously subjected to a distractive force such as byvacuum pressure. Injection into the breast may preferably beaccomplished with a syringe. The syringe may be a 3, 5 or 10 ml syringefitted with a 12 or 14 gauge cannula (i.e. approximately 2-3 mm indiameter). The injection may be into either one or both of a deepretroglandular space and/or a subcutaneous plane of the breast. Theinjections are preferably made in a three dimensional fanning pattern tomicro-weave the sample of fat in the breast. However, these injectionsare preferably not made into the breast parenchymal region or a breastglandular region. The preferred method of injecting the fat or dispersedtissue micro-droplet sample is to distribute them throughout the deepretroglandular space and/or the subcutaneous plane of the breast whileavoiding the breast parenchyma or breast gland region.

Following injection, a splint like device or bandage is preferablyapplied to the breast to immobilize the injected fat and facilitate theengraftment process. Alternatively, a distractive force such as vacuumpressure is again applied to the breast. The distractive force such asvacuum pressure may be first applied to the injected breast preferablyabout 12 to 18 hours after injection of the breast. For the two dayperiod following injection of fat into the breast the vacuum maypreferably be intermittently applied to the breast for at least about 6to 22 hours per day (i.e. 24 hour period) or for as long as possible.After this 2 day (i.e. 48 hour) period, the distractive force such asvacuum pressure may preferably be intermittently applied to the injectedbreast for at least about 10 hours per day during the entire postinjection time period of about 2 to 7 days.

This method may be applied when the subject is a human or, moreparticularly, a female human. It is further recognized that the entireprocedure comprising the steps of applying a distractive force to atleast one breast of a subject, removing a sample of fat from the subjectwith a syringe; injecting at least a portion of said sample of fat takenfrom the subject into said breast that was subjected to a vacuum with asyringe, and then applying a distractive force to the breast injectedwith the subject's fat can be repeated on one or more breasts of asubject. Repeating the procedure may be desirable as compared to asingle procedure in instances where greater degrees of breastaugmentation are sought.

Finally, alternative methods of obtaining dispersed soft tissue cellsamples suitable for injection into the subject are further disclosed.Such methods may be important in obtaining some substantial breastaugmentation when the subject has limited amounts of fat available forharvesting and injection. These methods might also be important when thetissue grafts have the potential to regenerate lost or scarred tissuenot only in the breast but in other organs and parts of the body. In oneembodiment, the fat sample may be obtained by removing fat from thesubject; processing the tissue to extract the preferred cellularcomponent such as adipocyte derived stem cells, and reinjecting theenriched tissue directly, or further propagating this component outsidethe subject, and then reinjecting it. In another embodiment, a sample offat may be obtained by removing a living cell or living cells from thesubject, propagating the cell or cells outside of the subject underconditions where the cell or cells will form a sample of fat; andcollecting the sample of fat thus formed. Also disclosed are methods ofobtaining the sample of fat from sources other than the subject. In oneembodiment of the invention, the source of the sample of fat may beobtained from another human, a human that is genetically related to thesubject, or a human that is a twin of the subject. Alternatively, thesample of fat may be obtained from a source that is a stem cell or cellsderived from a source other than the subject.

The above method of preparing the recipient site with a distractiveforce applied over a period of time long enough to substantially expandthe volume of the recipient tissue prior to grafting can also be appliedto graft augmentation of other parts of the body such as depressedscars, wrinkle lines or the subcutaneous fat atrophy that accompaniesaging. The method is also a useful way to augment the survival of fatgrafting to the buttocks. The method may also be used for thereconstruction of the breast after lumpectomy and radiation and aftermastectomy. The method can also be applied to other organs and parts ofthe body that have been lost to scarring, radiation, disease or agingand that could benefit from an infusion of stem cells or dispersedmicrografts of other cell types.

This method pertains to soft tissue three dimensional grafting ingeneral by providing a means of improving the success and engraftmentyield of the dispersed soft tissue cells by at least temporarypre-expansion of the recipient site utilizing distractive forces.

Further features and advantages of the present invention, as well as thestructure and operation of various embodiments of the present invention,are described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 illustrates the use of an external breast tissue expander (Brava®Breast Enhancement and Shaping System, Brava, LLC; Coconut Grove, Fla.);

FIG. 2 illustrates the three dimensional fanning pattern of injectionused to distribute the autologous fat grafts in a subject;

FIG. 3 illustrates magnetic resonance imaging results taken before and 3months following the breast augmentation procedure;

FIG. 4 illustrates a cross-sectional view of a woman's breast;

FIG. 5 illustrates an enlarged cross-sectional view of the subdermalperiglandular plane taken from the indicated portion of FIG. 4;

FIG. 6 illustrates the beneficial effect of preparation of the recipienttissue by the distractive force (external tissue expansion);

FIG. 7 shows a pattern of micrograft tissue graft dispersion in a threedimensional soft tissue block that is even and conducive to successfulengraftment. Extrapolating from the known two dimensional state, thisthree dimensional model illustrates that the limit of graft to recipientvolume imposed by crowding and revascularization has to remain below 1,even with the most even dispersion of the smallest micro-grafts.

FIG. 8 illustrates a conceptual three dimensional block of soft tissuerecipient, with FIG. 8 a illustrating the initial state, FIG. 8 billustrating the maximum amount of most evenly dispersed micrograftgrafts that can be successfully engrafted, FIG. 8 c illustrating thatattempts to stuff more grafts leads to necrosis because crowdinginterferes with revascularization of a significant portion of the graft,FIG. 8 d illustrating the original block of soft tissue recipient afterit has been subjected to expansive forces and increased in volume bydoubling each of its dimensions, and FIG. 8 e illustrating how moregrafts can now be successfully engrafted following expansion of therecipient block of soft tissue.

FIG. 9 shows the paradigm shift this invention has produced in the fieldof breast reconstruction in particular, and soft tissue regeneration ingeneral.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In practicing the embodiments of the method of this invention, apreferred method for applying distractive force such as vacuum pressureto the breast of said subject is by use of a device comprising a domewhich fits over at least one breast of the subject, a rim that sealssaid dome around said breast, and a pump for creating a vacuum aroundsaid breast. An external breast tissue expander (Brava® BreastEnhancement and Shaping System, Brava, LLC; Coconut Grove, Fla.)represents one preferred device for applying vacuum pressure to thebreast of the subject. Descriptions of various devices suitable forapplying vacuum pressure to the breast of a subject are described inU.S. Pat. Nos. 6,500,112, 5,676,634, 5,695,445, and 5,536,233 as well asin U.S. patent application Ser. No. 11/101,240, the disclosures of whichare incorporated herein by reference in their entirety. However, otherdevices that exert a distractive force on the breast may also be used inthe practice of this invention. For example, U.S. Pat. No. 6,699,176,incorporated herein by reference in its' entirety, discloses otherdevices that can exert a distractive force on the breast. One suchdevice comprises at least one dome for adhering to a patient's breast,where the dome includes a supporting frame that is inflatable to expandthe frame and induce a distractive force in the underlying breast. Oneskilled in the art would thus understand that the distractive force onthe breast may be exerted by either vacuum pressure, by mechanicalforces being applied, or by other means in accomplishing certain stepsof the method described herein. The distractive force may also beapplied internally, through tissue expanders as known in the art whichare inserted subcutaneously or with supporting frameworks that are madeto gradually expand, etc., all as may be known to those of skill in theart.

Without being limited by theory, and offered only as an observation, thesuccess of the method for breast augmentation by fat grafting disclosedherein may be due to one or more conditions or combinations ofconditions induced in the breast by the distractive force created by avacuum or by other methods. For example, pre-grafting expansion of thebreasts by subjecting them to a distractive force before introduction ofthe fat graft may increase the vascularity of the recipient site andthus result in improved quality of the graft recipient bed thusincreasing receptivity and retention (i.e. reduced graft failure).Similarly, pre-treatment of the breast with a distractive force (i.e.pre-grafting expansion of the breast) may provide for more periglandularor interstitial space for greater graft volume injection with lesscrowding of the injected fat graft droplets. This enlargement of thethree dimensional lattice improves the graft to recipient contactinterface for better revascularization of the injected fat. Post-graftinjection treatment of the breast with an immobilizing device or adistractive force may also hold up the grafts as a stent and promoterevascularization or other beneficial processes during the earlyengraftment period.

In practicing the invention, the distractive force is applied to thebreast prior to the initiation of fat grafting. In the examples providedherein, the distractive force is continuously applied for at least 24hours prior to fat grafting or injection. For about 28 days prior to theperiod of continuous distractive force application to the breast, anintermittent distractive force is applied to the breast for at least 10hours per day in the examples. Alternatively, higher distractive forcesintermittently applied over shorter periods of time might achieve thesame hypervascularity and interstitial space expansion effect. Inoutlining the time periods of distractive force application used in theexamples cited herein, one skilled in the art would clearly understandthat minor deviations from these exemplary time periods for applicationof the distractive force are not expected to significantly impact theresults obtained in practicing this invention. It is furthercontemplated that practitioners may adapt these time periods ofdistractive force application based on a variety of circumstancespertaining to each particular subject.

The amount of distractive force that is applied to the breast bothbefore and possibly after injection is desirably regulated. When thedistractive force is a vacuum, regulation of the vacuum within the domeis important to prevent contusions caused by rupturing capillariesadjacent the surface of the skin. Medical data suggest that thesecontusions will not occur if vacuum within the dome is maintained atless than about 20-25 mm Hg, at least on a continuous basis. Thus, thevacuum pump is desirably regulated to control the vacuum within the dometo within this limit presuming a protocol involving extended wearing atcontinuous pressure is followed. In addition, skin ulceration can occurif excessive contact pressures are applied thereto for any extended timeperiod. Medical data suggest that a contact pressure less than 20 mm Hgmay be applied indefinitely without such ulceration. However, contusionsmay occur due to positive contact pressures upon the skin at continuouspressures above this ulceration limit. Alternatively, higher pressurescould be intermittently applied for shorter periods of time over a fewweeks to achieve the same hypervascularity and expansion effect. Theinventor's invention contemplates use not only of one of the severaldistractive force application methodologies as recited herein but othersas well, subject to such further experimentation as would be well withinthe scope of the teaching and skill of those of ordinary skill in theart. For example, the inventor contemplates that cycled intermittedhigher vacuum pressures could be applied for shorter periods of time toachieve similar if not the same results.

Nonetheless, it is envisioned that vacuum pressures falling outside ofthe range of 20-25 mm Hg may also be employed in the practice of thisinvention. As discussed in U.S. patent application Ser. No. 11/101,240,incorporated herein by reference in it's entirety, approximately 20,000external breast tissue expander (Brava® Breast Enhancement and ShapingSystem, Brava, LLC; Coconut Grove, Fla.) devices have been soldworldwide as of the end of the year 2004, with approximately 10,000 ofthese sales being made in the U.S. alone. As part of these sales, thebuyers were provided medical advice through medical professionals anddata including their “wear” history was collected from approximately 78%of them. The recommended regimen required the user to wear the externalbreast tissue expander at least ten hours per day for ten consecutiveweeks. Pressures exerted within the bra ranged within 15 to 33 mm Hg asthe vacuum pump cycled off and on, with an average of approximately 22mm Hg. Users were also advised that wearing the external breast tissueexpander for longer time periods would result in greater increases inbreast size. As a result, it is believed that many users chose to wearthe device more than the ten hours per day and more than ten weeks.However, the recommended “wear” protocol was for ten hours per day forten full weeks. As in any recommended protocol, and especially oneextending for a significant time period, adjustments were made uponpatient request and upon patient initiated change without advice. Forsome users, requests were granted to increase the maximum pressure to 35and even 37 mm Hg. It is thus contemplated that under certaincircumstances, certain conditions, or during certain periods of time itmay be desirable to increase the amount of distractive force appliedbeyond the recommended range of 20 to 25 mm Hg in the practice of thisinvention.

The sample of fat for injection into the breast or breasts may be takenfrom any suitable region of the subject. Subjects are typically placedunder local tumescent anesthesia and sedation for the steps of theprocedure involving fat removal and fat injection. For example, the fatsamples may be harvested from an abdominal region, a thigh region, aflank region, a gluteal region, or a trochanter of said subject. Fattaken from multiple regions may be injected into a breast or breasts.Removal of fat is achieved by methods that minimize trauma and minimizemanipulation of the fat removed from the subject. In this regard, asyringe may be used to remove the fat. Manual removal of the fat with asmall syringe fitted with a fine diameter cannula is a preferred methodof obtaining the fat sample. One example of a preferred method ofobtaining the sample of fat is by aspirating the fat from the subjectwith a 10 ml syringe fitted with a 14 gauge cannula (i.e. approximately1-2 mm in diameter). The typically employed suction devices that areused in cosmetic surgery to remove fat (i.e. liposuction) may subjectthe fat to shear forces or other conditions that are less conducive tosuccessful grafting. However, it is recognized that mechanical devicesthat remove fat under reduced pressures and/or through fine diametercannulas may also be used to obtain a fat sample useful in the practiceof this invention.

Following removal and prior to injection, the harvested fat is thenseparated from any fluid such as serum or tumescent fluid that isassociated with the fat. Such separation may simply be accomplished byplacing the filled collection syringe in an upright position andallowing the fat and fluid to separate into two phases. The fluid phasecan then be simply expelled from the bottom of the syringe. Alternativemethods of separating fat and fluid such as centrifugation or filtrationare also contemplated.

The fat sample is then injected into the breast that was previouslysubjected to a distractive force. Injection into the breast may beaccomplished with a syringe. The syringe may be a 10 ml syringe fittedwith a 14 gauge cannula (i.e. approximately 1-2 mm in diameter).Injection may be into either one or both of a deep retroglandular spaceand/or a subcutaneous plane of the breast. The injections are made in athree dimensional fanning pattern to micro-weave the sample of fat inthe breast. FIG. 2 illustrates one arrangement of injection ports thatcan be used to inject the fat sample in a three dimensional fanningpattern. The injection ports are typically placed at regular intervalsaround both the base of the breast and around the nipple in a circularpattern. A frontal view of the breast post injection would thus show aninner circle of injection ports around the nipple and an outer circle ofinjection ports around the base of the breast. The cannula that is usedto distribute microdroplets or tiny droplets of fat is then inserted atvarious depths and angles to deliver a diffuse distribution of fat inthe desired regions of the breast. A microdroplet is typically betweenabout 25 ul (microliters) to 250 ul in volume. While it is anticipatedthat tiny droplets are desired, it is only known at present thatinsertion of large globules of fat need to be avoided. However,injections are preferably not made into a breast parenchyma region or abreast gland region. The preferred method of injecting the fat sample isto use multiple injection ports to distribute microdroplets of the fatsample throughout the deep retroglandular space and/or a subcutaneousplane of the breast while avoiding the breast parenchyma or breast glandregion. Avoidance of injection into the breast parenchyma or breastgland region may reduce the incidence graft failure and necrosis. Indescribing one illustrative pattern of injection ports, it is clearlyunderstood that any other pattern of injection ports that permits adiffuse distribution of the fat graft to the desired regions can be usedin the practice of this invention. For example, a grid pattern ofinjection ports or the use of curved injection cannulas through a morelimited number of injection ports (i.e. 1 or 2 sites only) may also beused.

Following injection of the fat into the breasts, a distractive forcemight be again applied to the breast or breasts. A preferred method forapplying distractive force such as vacuum pressure to the breast of saidsubject is by use of a device comprising a dome which fits over at leastone breast of the subject, a rim that seals said dome around saidbreast, and a pump for creating a vacuum around said breast.

In practicing the invention, the distractive force is possibly appliedfollowing the fat grafting or injection. In the examples providedherein, the distractive force is first applied about 12 to 18 hoursafter completion of fat injection. During the 48 hour period followingcompletion of fat injection, the distractive force is applied to theinjected breast as continuously as possible for at least about 6 to 22hours per day (i.e. 24 hour period) or for as long as possible. Afterthe 48 hour period following completion of fat injection, thedistractive force is intermittently applied to the injected breast forat least 10 hours per day for a period of about 2 to 7 days. Inoutlining the time periods of distractive force application used in theexamples cited herein, one skilled in the art would clearly understandthat minor deviations from these exemplary time periods for applicationof the distractive force are not expected to significantly impact theresults obtained in practicing this invention. It is furthercontemplated that practitioners may adapt these time periods ofdistractive force application based on a variety of circumstancespertaining to each particular subject.

This method may be applied when the subject is a human or, moreparticularly, a female human. This method may also be employed for othermedical or cosmetic needs or desires such as for example in the courseof augmenting the breasts of a human undergoing a sexual reversalprocedure wherein the breasts of a male human are first augmented byhormonal stimulation and/or surgical alteration to resemble the breastsof a female human.

Furthermore, this method might also offer an alternative to thereconstruction of the breast mound after lumpectomy, quadrantectomy,mastectomy, and radiation therapy. The current method of breastreconstruction consists of surgically inserting tissue expandersconsisting of silicone bags and gradually inflating them with injectablesaline over a period of weeks to expand and restore the skin envelope ofthe breast. The inflated expander creates a tissue void which needs tobe replaced by an implant in a subsequent surgical procedure. In theproposed alternate application, external expansion can be used togenerate the envelope and the recipient matrix space which will begradually filled with liposuctioned fat grafts. The external expansionwill also help generates the new blood vessels that can revascularizethe grafts. The grafting may be done in one or multiple sessions over aperiod of weeks to months. This method will gradually transfer fat fromother parts of the body and ensure the survival of that tissue torestore the breast mound (see FIG. 9).

In practicing this invention, the subject is advised to maintain theirbody weight, When the subject is of the female gender, the subject isfurther advised to take measures to avoid pregnancy during theprocedure.

The degree of breast augmentation obtained by performing this proceduremay be assessed by any number of methods. For example, breastaugmentation volume may be derived from the 3D (3 dimensional)volumetric reconstruction of the MRI scans taken prior to and followingthe procedure or method. Typically, a baseline MRI to determine aninitial pre-procedure volume is taken prior to initiating the first stepof the method (i.e. before applying a distractive force to theun-injected breast). Additional MRI is performed at 3 months and 6months post injection. A comparison of the breast volumes at 6 monthsrelative to those at 3 months post injection permits an assessment ofstability and long term graft survival result. Average augmentationvolumes at 6 months post-procedure may be about 170 ml per breast andmay range from about 90 ml to 220 ml. In some instances, the degree ofbreast augmentation attained may be limited by the amount of autologousfat that can be harvested from the subject.

In view of the limits to breast augmentation potentially imposed incertain instances by the amount of autologous fat that can be harvestedfrom a subject, it is also contemplated that the fat sample can beobtained by methods other than direct removal and immediate reinjectioninto the subject. For example, the fat sample may be obtained byremoving fat from the subject; propagating this removed fat outside ofthe subject, and collecting the fat sample that was expanded throughpropagation. Propagation in this context may be in an in vitro culturesystem specifically adapted to growth and expansion of fat.Alternatively, a sample of fat may be obtained by removing a living cellor living cells from the subject, propagating the cell or cells outsideof the subject under conditions where the cell or cells will form asample of fat; and collecting the sample of fat thus formed. In thecontext of a living cell or cell removed from the host, propagation ofthe cell or cells may be in an in vitro culture system that promotes thedifferentiation of a non-fat cell or fat cell progenitor cell into a fatcell. The non-fat cell may be a stem cell. One skilled in the art willrecognize that a fat cell or fat cell progenitor may also be referred toas an adipose cell or adipose cell progenitor. Under these conditionsthe non-fat cell or fat cell progenitor cells may expand by celldivision prior to becoming a fat cell. In the context of a living cellor cell removed from the host, propagation of the cell or cells may bein an in vitro culture system that promotes the expansion of a fullydifferentiated fat or adipose cell. In this case, expansion of the fullydifferentiated cell may occur by accumulation of fat within the cellrather than by cell division. Finally, it is contemplated that theliving cell or cells removed from the host under conditions where somecombination of cell division and cell expansion results in the formationof a fat sample suitable for reinjection into the subject. Variousmethods and devices suitable for propagating cells outside of a sourceorganism that may include a subject or another human are disclosed inU.S. Pat. No. 6,238,908, the contents of which are incorporated hereinby reference in their entirety.

Also contemplated are methods whereby the engrafted stem cells areinduced to regenerate lost tissue other than fat tissue in organs otherthan the breast.

Also contemplated are methods of obtaining the sample of fat fromsources other than the subject. For example, the source of the sample offat may be obtained from another human, a human that is geneticallyrelated to the subject, or a human that is a twin of the subject.Alternatively, the sample of fat may be obtained from a source that is astem cell or cells derived from source other than the subject. The stemcell or cells may be propagated outside of the subject under conditionswhere said stem cell or cells form a sample of fat as described in theforegoing discussion of conditions used to obtain fat samples from acell or cells derived from the subject.

It is further recognized that the entire procedure that comprises thesteps of applying a distractive force to at least one breast of asubject, removing a sample of fat from the subject; injecting at least aportion of said sample of fat taken from the subject into said breastthat was subjected to a vacuum, and then applying a distractive force tothe breast injected with the subject's fat can be repeated on one ormore breasts of a subject. Repeating the procedure may be desirable ininstances where greater degrees of breast augmentation are sought.

While breast augmentation and reconstruction with liposuctioned fat isthe example used here with a potential immediate clinical application,it would be understood by those of ordinary skill in the art that thisinvention pertains to three dimensional soft tissue grafting in general.The at least temporary pre-expansion of the recipient site by utilizingdistractive forces improves the success and engraftment yield of thedispersed soft tissue cell grafts.

EXAMPLE

The following example of the invention is provided to illustrate themethods of this invention. Those skilled in the art will recognize thatvarious modifications of the methods described herein may be madewithout departing from the gist of the invention.

Example One

After Institutional Review Board approval and informed consent of thesubjects, 28 women underwent 30 fat grafting procedures on 55 breasts.This was achieved by performing one unilateral post cancerreconstruction with two grafting procedures, 24 bilateral primary breastaugmentations with one grafting procedure and two bilateral primarybreast augmentations with two grafting procedures. Following a baselineMRI (Magnetic Resonance Imaging) of the breast, an external breasttissue expander (Brava® Breast Enhancement and Shaping System, Brava,LLC; Coconut Grove, Fla.) was worn 10 hours per day for four weeks bythe subjects. FIG. 1 shows a subject wearing an external breast tissueexpander (Brava® Breast Enhancement and Shaping System, Brava, LLC;Coconut Grove, Fla.). For the 24 hour period immediately preceding theprocedure, the subjects were asked to continuously use the externalbreast tissue expander and to come to the procedure still wearing theexpander. This last pre-procedure step induces a marked temporaryexpansion of the subcutaneous plane creating more recipient space forthe grafts. Under local tumescent anesthesia and sedation, using 12-14 G(gauge; 2-3 mm diameter) cannulas on 10-60 ml syringes, between 90 to380 ml of fat per side was aspirated from the thighs, the flanks and thetrochanters. The graft amount was limited by the ease of harvesting. Thefat was separated from the serum and the tumescent fluid by decantingfor a few minutes on a rack or by gently centrifuging it with a handoperated centrifuge. It was then re-injected into the ipsilateral breastthrough a multitude of needle puncture sites in a three dimensionalfanning pattern, micro-weaving the grafts using 3-10 ml syringes and a12-14 G (gauge; 2-3 mm diameter) re-injection cannulas. The threedimensional fanning pattern of injection is shown in FIG. 2. In 11procedures, some of the grafts were deliberately placed intraglandular.In the remaining procedures, the injection was limited to thesubcutaneous plane and to the deep retroglandular space with strictavoidance of the breast parenchyma. Twelve hours post procedure, thesubjects were asked to place the external breast tissue expander on andto keep wearing it as much as possible for the next few days to hold upthe grafts as a stent during the revascularization and early engraftmentperiod. At three months post-procedure, a second MRI was obtained, andat 6 months post-procedure, a third MRI and a mammogram were obtained.Breast augmentation volume was derived from the 3D volumetricreconstruction of the MRI scans. FIG. 3 shows a comparison of thepre-procedure and 3 month post-procedure MRI scans of a subject.

Except for some temporary bruising and superficial skin blisters thathealed uneventfully, there were no significant complications and allwomen returned to their normal activities within 3 to 4 dayspost-procedure. The patients were all pleased with the enlargement andimproved appearance of their breasts and liposuctioned donor site. Therewere no palpable breast masses or nodules. The breast volume increaseretained at 3 months remained constant at 6 months indicating permanentgraft survival (3- vs. 6-month, p>0.2; paired t-test). The averageaugmentation volume at 6 months was 195 ml per breast and ranged from100 ml to 320 ml. Graft survival averaged 90% and ranged from 120% (someof the women gained weight and in some cases the original grafted cellsgrew) to 70%. Though the numbers did not achieve statisticalsignificance, graft survival was lower in the group where some of thegrafts were injected intraglandular. More importantly, there was no fatnecrosis in the group with strict periglandular grafting compared to theisolated intra-parenchymal foci of fat necrosis in the breasts that alsoreceived intraglandular grafts (0/14 vs. 5/11; p=0.0087, Fisher Exacttest).

The inventor has some recent experimental animal (rat) data whichdemonstrates that the application of a distractive force over a periodas short as two weeks is very angiogenic. By placing vacuum domes on theback of rats and applying vacuum over a period of two weeks, theinventor observed the formation of a very marked vascular and capillarynetwork inside the domes that had vacuum applied. This observed intenseneovascular response with the generation of a large new vascularnetwork. creates an ideal condition for successful graft take andsurvival. This angiogenic or neovascularization effect of pre-graftingexpansion is one of the main advantages of this technique.

Yet another advantage of pre-grafting expansion is that it opens up therecipient space to allow more grafts placement with less crowding. It isimportant to avoid grafting in the breast parenchymal glandular tissueitself and the inventor has shown from his experimental work that thebest recipient site is the subdermal periglandular plane. (In the caseswhere some grafts were deliberately placed in the glandular tissue, alower overall percentage graft take and more fat necrosis was found tooccur). Pre-expansion enlarges that preferred recipient plane to allowfor more grafts there. It is well recognized by experts in the art ofplastic surgery and grafting that improving the surface contact areainterface between the graft and the recipient bed improves graft takeand survival. The best graft survival is thought to occur when verysmall pieces (microdroplets) of fat are completely surrounded by a wellvascularized recipient bed. These droplets should be separated from eachother by recipient tissue and any crowding or graft to graft contact isthought to be detrimental. Hence the great beneficial effect of thispre-grafting expansion. The inventor generates the three dimensionalnetwork lattice and space to disperse more grafts with less crowding.The tissue may be likened to a sponge, and expansion enlarges thatsponge allowing it to absorb many more “microdroplets” with betterdispersion and less crowding. See FIGS. 4-6.

In summary, the inventor believes that he has with just his present workproven at least four major beneficial effects of pre-grafting expansion:1—generation of new blood vessels or angiogenesis, which is well knownto improve graft take, 2—generation of additional recipient space andthree dimensional framework which makes room for more grafts to havebetter dispersion and less crowding, factors that optimize the crucialgraft-to-recipient interface, 3—generation of an extracellular matrixfluid that is rich in growth factors and agents that render the spacemore fertile for graft success, and 4—a post operative stent likeimmobilization device that secures engraftment by allowing vascularingrowth

In view of the foregoing, it will be seen that the several advantages ofthe various aspects of the invention are achieved and attained.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. Thus, the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

1) A method for augmenting a soft tissue site of a subject, comprisingthe steps of: a) applying a distractive force to said soft tissue site;b) obtaining a sample of fat; and c) injecting at least a portion ofsaid sample of fat from step (b) into said soft tissue site that wassubjected to a distractive force; thereby augmenting the soft tissuesite of said subject. 2) The method of claim 1, wherein the distractiveforce is continuously applied in step (a) for at least 24 hours prior toperforming step (c). 3) The method of claim 2, wherein the distractiveforce is intermittently applied in step (a) for at least 10 hours perday for about 28 days prior to performing step (c). 4) The method ofclaim 1, wherein the distractive force of step (a) is an externallyapplied mechanical force. 5) The method of claim 4, wherein said softtissue site is at least one human breast and the distractive force isapplied in step (a) with a device comprising a dome which fits over saidat least one breast, a rim that seals said dome around said breast, anda pump for creating a vacuum within said dome. 6) The method of claim 5,wherein said rim has a sticky surface adapted to adhere to the patient'sskin to thereby create and hold a pressure seal between said dome andsaid patient. 7) The method of claim 5, wherein said pump is a manualpump. 8) The method of claim 5, wherein said pump further comprises anindependent power source and a pressure sensor. 9) The method of claim8, wherein said pump is continuously engaged with said device tomaintain a constant vacuum pressure inside said dome. 10) The method ofclaim 9, wherein said constant vacuum pressure is maintained betweenabout 20 to 25 mm of Hg. 11) The method of claim 1, wherein steps (a)and (c) are performed contemporaneously on two breasts of a humansubject. 12) The method of claim 11, wherein both breasts are subjectedto a similar vacuum pressure with a device comprising two domes whichfit over said breasts, two rims that seal said domes around saidbreasts, a pump, and a conduit that places said pump in fluidcommunication with each of said domes. 13) The method of claim 12,wherein one breast is subjected to a different vacuum pressure than theother breast in step (a) with a device comprising two domes which fitover the breasts of said subject, a rim that seals each of said domesaround each of said breasts, and a separate pump for each of said domes.14) The method of claim 1, wherein said distractive force is appliedwith a device which mechanically attaches to the soft tissue site andinduces a distractive force in the underlying soft tissue site. 15) Themethod of claim 14 wherein said distractive force is applied with adevice positioned internally to said soft tissue site. 16) The method ofclaim 1, wherein said sample of fat is removed with a syringe from atleast one of an abdominal region, a thigh region, a flank region, agluteal region, or a trochanter of said subject. 17) The method of claim16, wherein said syringe is approximately a 10 ml syringe fitted with a14 gauge cannula. 18) The method of claim 1, further comprisingseparating said sample of fat from serum and tumescent fluid prior tostep (c). 19) The method of claim 18, wherein said sample of fat isseparated from serum and tumescent fluid by decanting the fluid from thefat. 20) The method of claim 1, wherein said soft tissue is injectedwith said sample of fat in step (c) with a syringe. 21) The method ofclaim 20, wherein said syringe is a 10-50 ml syringe fitted with a 14gauge cannula (2-3 mm). 22) The method of claim 1, wherein saidinjections of step (c) are into a subcutaneous plane of a breast. 23)The method of claim 1, wherein said injections of step (c) are into adeep retroglandular space of a breast. 24) The method of claim 1,wherein said injections of step (c) are into both a deep retroglandularspace and a subcutaneous plane of a breast. 25) The method of claim 1,wherein said injections of step (c) are made in a three dimensionalfanning pattern to micro-weave said sample of fat in a breast. 26) Themethod of claim 25, wherein said injections of step (c) are made inthose regions of a breast not including a breast parenchyma region. 27)The method of claim 1, wherein said injections of step (c) are made byinjecting microdroplets of said fat sample throughout a breast. 28) Themethod of claim 1, wherein said soft tissue comprises at least onebreast and further comprising applying a distractive force to saidbreast after it is injected at about 12 to 18 hours after completion ofstep (c). 29) The method of claim 28, wherein the distractive force isintermittently applied to said breast for at least about 6 to 22 hoursper day for about 2 days after step (c). 30) The method of claim 29,wherein the distractive force is intermittently applied to said breastfor at least about 10 hours per day for about 2 to 7 days after step(c). 31) A method of improving the survival of a fat graft into atargeted soft tissue site including applying a distractive force to saidsoft tissue site prior to the fat graft. 32) The method of claim 31wherein the step of applying the distractive force includes applying atleast intermittently a vacuum to said soft tissue site for at least 8hours prior to grafting. 33) The method of claim 1, wherein said sampleof fat is obtained by a method comprising the steps of: a) removing fatfrom said subject; b) propagating said fat from step (a) outside of thesubject; c) and collecting the sample of fat formed in step (b). 34) Themethod of claim 1, wherein said sample of fat is obtained by a methodcomprising the steps of: a) removing at least one living cell from saidsubject; b) propagating said cell or cells from step (a) outside of thesubject under conditions where said cell or cells form a sample of fat;c) and collecting the sample of fat formed in step (b). 35) The methodof claim 1, wherein said sample of fat is obtained from a source otherthan said subject. 36) The method of claim 35, wherein said source is ahuman. 37) The method of claim 36, wherein said human is geneticallyrelated to said subject. 38) The method of claim 36, wherein said humanis a twin of the subject. 39) The method of claim 1, wherein said sourceis a stem cell or cells. 40) The method of claim 39, wherein said stemcell or cells are propagated outside of the subject under conditionswhere said stem cell or cells form a sample of fat. 41) A method ofpreparing a soft tissue site for receiving a fat graft comprisingidentifying a portion of soft tissue targeted for receiving a fat graft,and applying a distractive force to said targeted soft tissue site foran extended time period prior to injecting a fat graft into said softtissue site. 42) The method of claim 41 wherein applying saiddistractive force comprises applying an external mechanical force. 43)The method of claim 42 wherein applying said external mechanical forcecomprises applying a vacuum at least intermittently to said soft tissuesite. 44) The method of claim 43 wherein applying said vacuum includesapplying a vacuum for periods of time within about 10 hours per day forabout four weeks prior to the fat grafting. 45) The method of claim 43wherein applying said external mechanical force includes applying thedistractive force substantially continuously for the 24 hours nextpreceding the injection of fat. 46) The method of claim 41 wherein thetargeted soft tissue site includes a human breast, and applying adistractive force includes externally applying a vacuum.